Maple syrup urine disease caused by a partial deletion in the inner E2 core domain of the branched chain alpha-keto acid dehydrogenase complex due to aberrant splicing. A single base deletion at a 5'-splice donor site of an intron of the E2 gene disrupts the consensus sequence in this region.

Abstract

We have studied the molecular bases of maple syrup urine disease by analyzing the activity, subunit structure, mRNA sequence, and the genome of the affected enzyme. The branched chain a-keto acid dehydrogenase (BCKDH) activity in the patient was 4.2-4.5% of the control level. Immunoblot analysis revealed that the E2 subunit of BCKDH (M, 52,000) was absent and another protein band with an M, of 49,000 was present. We amplified the cDNA of the E2 subunit obtained from the patient's cell using the polymerase chain reaction method, then sequenced the amplified cDNA, in which a 78-bp deletion was identified. The consanguineous parents and a sister had two species of mRNA; the one corresponding to the normal E2 subunit and the other with a 78-bp deletion, whereas findings in a brother were normal. The molecular size of the translation products as deduced from the abnormal mRNA sequence was compatible with an abnormal protein band (M, 49,000) detected in the patient's cells by immunoblot analysis.Analysis of genomic DNA of BCKDH-E2 subunit revealed that the 78-bp deletion in the mRNA was caused by an exon skipping due to a single base deletion in the 5'-splice donor site. As a result ofthe mutation, part ofthe inner E2 core domain was omitted. The specified region of the inner E2 core domain was highly homologous to the region of the E2 subunit of pyruvate dehydrogenase and a-ketoglutalate dehydrogenase. These observations imply the biological importance of the region in the inner E2 core domain of BCKDH to maintain normal function of the activity.

Identification of twelve novel mutations in patients with classic and variant forms of maple syrup urine disease

“…This specific region of the inner E2 core domain indicates a strong species conservation and is highly homologous to the region of the E2 subunits of the pyruvate and α-ketoglutarate dehydrogenase and thus seems biologically important to maintain a normal function of the E2-protein (Russell and Guest, 1991). The known splice site mutation E2-c.1017+1delG, a single base deletion in the 5'-splice donor site, also leads to deletion of exon 8 (Mitsubuchi et al, 1991). The identified novel nonsense mutations (E1β-K241X, E1β-R285X), deletions (E1β-c.595_596delAG, E1β-c.937delG) and the duplication (E1β-c.163_166dupACTT) in the E1β-and E2-subunits can easily be predicted to impair the BCKD complex activity since they all lead to premature termination codons.…”